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Francesca Rochais, Aniella Abi-Gerges, Kathleen Horner, Florence Lefebvre,
Dermot Cooper, Marco Conti, Rodolphe Fischmeister, Grégoire Vandecasteele
To cite this version:
Francesca Rochais, Aniella Abi-Gerges, Kathleen Horner, Florence Lefebvre, Dermot Cooper, et al.. A Specific Pattern of Phosphodiesterases Controls the cAMP Signals Generated by Dif-ferent Gs-Coupled Receptors in Adult Rat Ventricular Myocytes.: PDEs and hormone specific cAMP signals. Circulation Research, American Heart Association, 2006, 98 (8), pp.1081-8. �10.1161/01.RES.0000218493.09370.8e�. �inserm-00000047�
This is an un-copyedited author manuscript that was accepted for publication in Circulation Research, copyright The American Heart Association. This may not be duplicated or reproduced, other than for personal use or within the “Fair Use of Copyrighted Materials” (section 107, title 17, U.S. Code) without prior permission of the copyright owner, The American Heart Association. The final copyedited article, which is the version of record, can be found at http://circres.ahajournals.org/. The American Heart Association disclaims any responsibility or liability for errors or omissions in this version of the manuscript or in any version derived from it.
A Specific Pattern of Phosphodiesterases Controls the cAMP Signals
Generated by Different G
s-Coupled Receptors in Adult Rat Ventricular
Myocytes
Francesca Rochais, Aniella Abi-Gerges, Kathleen Horner, Florence Lefebvre, Dermot M. F Cooper, Marco Conti, Rodolphe Fischmeister and Grégoire Vandecasteele
From INSERM U-769 (F.R.,A.A.-G.,F.L., R.F., G.V.), Châtenay-Malabry, F-92296 France; Université Paris-Sud (F.R.,A.A.-G.,F.L., R.F., G.V.), Faculté de Pharmacie, Châtenay- Malabry, F-92296 France; Division of Reproductive Biology (K.H., M.C.), Department of Gynecology and Obstetrics, Stanford University, Stanford, USA; Department of
Pharmacology (D.M.F.C.), University of Cambridge, Cambridge, UK.
running title : PDEs and hormone specific cAMP signals Correspondence to: Dr. Rodolphe FISCHMEISTER INSERM U-769 Université de Paris-Sud Faculté de Pharmacie 5, Rue J.-B. Clément F-92296 Châtenay-Malabry Cedex France Tel. 33 1 46 83 57 57 Fax 33 1 46 83 54 75 E-mail: fisch@vjf.inserm.fr
Subject codes: [152] Ion channels/membrane transport, [138] Cell signalling/signal transduction, [85] Autonomic, reflex, and neurohumoral control of circulation
Abstract—Compartmentation of cAMP is thought to generate the specificity of Gs-coupled
receptors action in cardiac myocytes, with phosphodiesterases (PDEs) playing a major role in this process by preventing cAMP diffusion. We have tested this hypothesis in adult rat ventricular myocytes (ARVMs) by characterizing PDEs involved in the regulation of cAMP
signals and L-type Ca2+ current (ICa,L) upon stimulation with β1-adrenergic receptors (β1-AR),
β2-adrenergic receptors (β2-AR), glucagon receptors (Glu-R) and prostaglandin E1 receptors
(PGE1-R). All receptors but PGE1-R increased total cAMP, and inhibition of PDEs with
IBMX strongly potentiated these responses. When monitored in single cells by high affinity
cyclic nucleotide-gated (CNG) channels, stimulation of β1-AR and Glu-R increased cAMP,
whereas β2-AR and PGE1-R had no detectable effect. Selective inhibition of PDE3 by
cilostamide and PDE4 by Ro 20-1724 potentiated β1-AR cAMP signals, whereas Glu-R
cAMP was augmented only by PD4 inhibition. PGE1-R and β2-AR generated substantial
cAMP increases only when PDE3 and PDE4 were blocked. For all receptors except PGE1-R,
the measurements of ICa,L closely matched the ones obtained with CNG channels. Indeed,
PDE3 and PDE4 controlled β1-AR and β2-AR regulation of ICa,L, while only PDE4 controlled
Glu-R regulation of ICa,L thus demonstrating that receptor-PDE coupling has functional
implications downstream of cAMP. PGE1 had no effect on ICa,L even after blockade of PDE3
or PDE4, suggesting that other mechanisms prevent cAMP produced by PGE1 to diffuse to
L-type Ca2+ channels. These results identify specific functional coupling of individual PDE
families to Gs-coupled receptors as a major mechanism enabling cardiac cells to generate
heterogeneous cAMP signals in response to different hormones.
Key words: cAMP ■ heart ■ G protein-coupled receptor ■ phosphodiesterase
___________________________________________________________________________
Introduction
Cardiac myocytes express a number of Gs-coupled receptors (GsPCRs) that raise
intracellular cAMP levels and activate cAMP-dependent protein kinase (PKA) but exert
different downstream effects. For instance, β1-AR stimulation produces a major and sustained
increase in force of contraction, accelerates relaxation, and stimulates glycogen
phosphorylase.1 β2-AR stimulation also increases contractile force but does not activate
glycogen phosphorylase2 and does not accelerate relaxation1,3 (but see Bartel et al. (2003)2);
Glu-R stimulation activates phosphorylase and exerts positive inotropic and lusitropic effects,
but the contractile effects fade with time.4 Finally, PGE-1 has no effect on contractile activity
or glycogen metabolism.5,6
Such observations led to the proposal that activation of different GsPCRs results in the
accumulation of cAMP and phosphorylation of hormone target proteins in distinct
compartments.7 The discovery of A-kinase anchoring proteins, responsible for the subcellular
distribution of particulate PKA,8 and the development of new methodologies allowing to track
local cAMP in living cells have provided additional support for this concept.9-15
Because cAMP is a small and readily diffusible molecule, cAMP compartments can only exist under a restrictive set of conditions for the localization and activity of the different
components of the cAMP signaling cascade, namely GsPCRs, adenylyl cyclases, PKA and the
cAMP phosphodiesterases (PDEs). Cardiac PDEs fall into four families: PDE1, which is
activated by Ca2+/calmodulin; PDE2, which is stimulated by cGMP; PDE3, which is inhibited
by cGMP; and PDE4. While PDE1 and PDE2 can hydrolyze both cAMP and cGMP, PDE3 preferentially hydrolyzes cAMP and PDE4 is specific for cAMP. Until now, only a limited number of studies have addressed directly the participation of PDEs to cAMP
9,10,12,15,16
ventricular myocytes (ARVMs) to show that β-adrenergic cAMP signals are localized by
PKA-mediated activation of PDE3 and/or PDE4.13 However, PDE regulation of other
hormonal cAMP signals is presently unknown. In the following, we address this point by
comparing the cAMP signals generated by β1-AR, β2-AR, Glu-R and PGE1-R, their regulation
by individual PDE families, and the functional consequences on L-type Ca2+ channels.
Materials and Methods
Detailed methods are included in the online-only Data Supplement to this article, which is
available at http://www.circres.ahajournals.org.
Total cAMP determination - cAMP was measured by RIA after acetylation of the samples.20,21
PDE Assay- PDE activity was measured according to a modification of the two-step assay
procedure method described by Thompson & Appleman17 with 1 µM cAMP and 105 cpm
[3H]-cAMP, as detailed previously.18
Electrophysiological Experiments - The whole-cell configuration of the patch-clamp
technique was used to record the L-type calcium current (ICa,L) and the CNG current (ICNG).
The extracellular Cs+-Ringer solution contained 1.8 mM [Ca2+] and 1.8 mM [Mg2+] for ICa,L
recordings and nominal [Ca2+] and [Mg2+] plus 1µmol/L nifedipine for ICNG recordings. Patch
pipettes were filled with a Cs+-solution.
Data Analysis - ICa,L amplitude was measured as the difference between the peak inward
current and the current at the end of the 400-ms duration pulse.19 ICNG amplitude was
measured at the end of the 200 ms pulse. Capacitance and leak currents were not
compensated. In 141 ARVMs, mean capacitance was 160.5 ± 3.9 pF. ICNG density was
calculated for each experiment as the ratio of current amplitude to cell capacitance. All the
data are expressed as mean ± S.E.M. When appropriate, the Student’s t-test was used for
statistical evaluation.
Results
PDE activities in ARVMs
Figure 1 summarizes experiments where the relative activity of PDE2, PDE3 and PDE4 in quiescent ARVMs was determined. Total cAMP hydrolytic activity was on average 109.8±16.6 pmoles/min/mg protein (n=4) and was mainly due to PDE3 and PDE4, which represented 31% and 38% of the total, respectively. Under these assay conditions, PDE2 activity was minimal (Fig. 1). Immunoprecipitation experiments showed that PDE4 activity is contributed by PDE4A, PDE4B and PDE4D variants, while the PDE3A form is the predominant PDE3 expressed (data not shown). The relative activity of individual PDE families did not vary significantly after 24 h and 48 h of culture (data not shown). Moreover, the ratio of PDE3/PDE4 was similar to that measured in extracts of adult rat ventricle (data not shown). Thus, cultured ARVMs closely resemble the in vivo pattern of PDE expression.
(Figure 1 near here)
Total cAMP production in response to different GsPCRs in ARVMs
We next determined whether stimulation of different GsPCRs increased total cAMP in
ARVMs (Fig. 2). We used a combination of isoprenaline (ISO, 5 µmol/L) and the β2-AR
antagonist ICI 118551 (ICI, 1 µmol/L) to stimulate β1-AR, a combination of ISO (5 µmol/L)
in the presence of the β1-AR antagonist CGP 20712A (CGP, 1 µmol/L) to stimulate the β2
-AR, glucagon (Glu, 1 µmol/L) to stimulate Glu-R, and PGE1 (1 µmol/L) to stimulate PGE1-R.
Basal cAMP was more than doubled by ISO + ICI and Glu, and increased by ≈70% by ISO +
CGP. PGE1 had no effect on cAMP content under these experimental conditions. A 15 min
incubation with the broad spectrum PDE inhibitor IBMX (100 µmol/L) increased the total cAMP content by about two-fold and dramatically potentiated the response to all agonists
except PGE1. The effect of ISO + ICI and Glu were multiplied by 8 and 7, respectively, while
that of ISO + CGP was increased 3-fold. Cyclic AMP content after PGE1 + IBMX was
slightly increased when compared to IBMX alone although it did not reach statistical significance.
(Figure 2 near here)
Real-time monitoring of subsarcolemmal cAMP signals elicited by different GsPCRs in ARVMs
To further compare cAMP signals elicited by the different receptors, we used CNG channels that allow direct monitoring of cAMP variations beneath the plasma membrane in intact
ARVMs.13 Figure 3 summarizes the results obtained with two CNGA2 mutants which differ
in their sensitivity to cAMP, E583M (EC50 for cAMP ≈10 µmol/L) and C460W/E583M (EC50
for cAMP ≈1 µmol/L).20 As depicted in Fig. 3A, activation of β
1-AR by a combination of ISO
(5 µmol/L) and ICI (1 µmol/L) increased basal ICNG density ≈3-fold, whereas specific
activation of β2-AR by ISO (5 µmol/L) plus CGP (1 µmol/L) had no effect. Similarly,
activation of Glu-R by Glu (1 µmol/L) or PGE1-R by PGE1 (1 µmol/L) failed to activate ICNG
(Fig. 3A). These results indicate that cAMP concentration was below the activation threshold
of E583M CNGA2 upon stimulation of β2-AR, Glu-R and PGE1-R. When using the
C460W/E583M mutant (Fig. 3B), activation of β1-AR increased basal ICNG density 6-fold.
This effect was significantly higher (p<0.01) than that obtained in cells expressing E583M CNGA2, as expected from the relative affinities of the two channels toward cAMP.
Interestingly, application of Glu now activated ICNG by approximately 5-fold, but specific
C460W/E583M CNGA2 allowed the detection of subsarcolemmal cAMP generated by
activation of Glu-R but not by activation of β2-AR or PGE1-R.
(Figure 3 near here)
Regulation of subsarcolemmal cAMP signals by PDEs
The above results suggest that cAMP signals elicited by the four different GsPCRs at the
plasma membrane are not identical. We next investigated the contribution of PDEs to such specificity. Both CNGA2 mutants were used with essentially similar results, therefore the low affinity E583M channel data are presented as supplementary Figures I and II. Preliminary
experiments showed that IBMX (100 µmol/L) had no significant effect on ICNG from
myocytes expressing E583M/C460W (n=6, data not shown). Figure 4A shows a typical
experiment in which the amplitude of ICNG is depicted as a function of time, thus providing an
instant readout of subsarcolemmal cAMP level in a single cardiac myocyte. β1-AR
stimulation with ISO (5 µmol/L) + ICI (1 µmol/L) induced a substantial increase of ICNG,
which was strongly potentiated by selective PDE3 inhibition with cilostamide (Cil, 1 µmol/L) or selective PDE4 inhibition with Ro 20-1724 (Ro, 10 µmol/L). Concomitant inhibition of all
PDEs with IBMX (100 µmol/L) resulted in a similar potentiation of β1-AR stimulation to that
obtained with selective PDE4 inhibition. At the end of each experiment, the cell was challenged with a saturating concentration (100 µmol/L) of the forskolin analog L-858051
(L-85) to determine the maximal ICNG response. In Figures 4C and 4D, we investigated the role
of PDEs in shaping the β2-AR cAMP response. As seen before, application of ISO (5 µmol/L)
+ CGP (1 µmol/L) had no effect on ICNG. Selective inhibition of PDE3 during β2-AR
stimulation with ISO + CGP induced a small and transient increase of ICNG. A somewhat
stronger effect was observed upon PDE4 inhibition, although it was also transient (Fig. 4C).
In contrast, concomitant inhibition of PDE3 and PDE4 unmasked a robust and sustained
cAMP increase during β2-AR stimulation. These results indicate that PDE3 and PDE4
control cAMP elicited by β1-AR and β2-AR. Both PDEs are necessary to circumvent the
cAMP rise elicited by β1-AR, while either PDE3 or PDE4 individually are sufficient to
prevent cAMP to rise beneath membrane upon β2-AR activation.
(Figure 4 near here)
Figure 5A shows that inhibition of PDE3 had little effect on ICNG augmented by 1
µmol/L Glu while a selective inhibition of PDE4 by Ro produced a strong stimulation of the current. Concomitant inhibition of both PDE3 and PDE4 by Cil + Ro, or of all PDEs by IBMX, resulted in a comparable effect as inhibition of PDE4 alone (Fig. 5A and 5B). These results indicate that Glu-R cAMP signals at the membrane are exclusively controlled by
PDE4. Although no increase in cAMP with PGE1 could be detected before, we tested whether
PDE inhibition could reveal a PGE1 effect on ICNG. As shown in Fig. 5C, application of 1
µmol/L PGE1 did not affect ICNG. However, in the continuous presence of PGE1, simultaneous
inhibition of PDE3 and PDE4 (by Cil + Ro) clearly activated the CNG current. IBMX was more efficient than Cil + Ro, indicating that other PDEs besides PDE3 and PDE4 might play
a role under these conditions (Fig. 5C). Thus, similarly to β2-AR, cAMP mobilization by
PGE1-R needs concomitant inhibition of PDE3 and PDE4 to be detected at the membrane.
However, in contrast to β2-AR, other PDEs besides PDE3 and PDE4 regulate PGE1-R cAMP
signals in cardiac cells.
(Figure 5 near here)
Regulation of the L-type Ca2+ current by GsPCRs and PDEs
Collectively, the above results show that different PDEs are involved in the regulation of cAMP generated by distinct GsPCRs in cardiac myocytes. In order to evaluate the functional implication of these findings, we examined the regulation of a major sarcolemmal cAMP
target, the L-type Ca2+ current (I
Ca,L), by GsPCRs and PDEs. The experiments were performed
at 24 h, as with CNG channels experiments. Selective inhibition of PDE3 by Cil (1 µmol/L)
or PDE4 by Ro (10 µmol/L) had no effect on basal ICa,L (data not shown). As the
concentration of ISO (5 µmol/L) used in the β1-AR stimulation of ICNG produced a maximal
stimulation of ICa,L (121.3±22.2% over control, n=9, data not shown), a lower dose of ISO (1
nmol/L, with 1 µmol/L ICI) was used in subsequent experiments in order to assess the
contribution of PDE isoforms to the β1-AR regulation of ICa,L. In these conditions, selective
inhibition of PDE3 by Cil or PDE4 by Ro resulted in a marked potentiation of the
prestimulated ICa,L (Fig. 6A and 6B). As shown in Fig. 6C and 6D, β2-AR stimulation with
ISO (5 µmol/L) and CGP (1 µmol/L) produced a small but significant stimulation of ICa,L.
This effect could be further potentiated by PDE3 inhibition with Cil and more markedly by PDE4 blockade with Ro. These results indicate that PDE3, and more prominently PDE4, are
integral components of ICa,L regulation by β1-AR and β2-AR. In another series of experiments,
the regulation of ICa,L by Glu was investigated. At high (1 µmol/L) concentration, Glu
maximally stimulated ICa,L and addition of PDE inhibitors had no effect (data not shown). At
the concentration of 10 nmol/L, Glu slightly decreased ICa,L and PDE3 inhibition by Cil had
no effect. However, application of Ro to block PDE4 clearly increased ICa,L (Fig. 7A and 7B).
Finally, as shown in Fig. 7C and 7D, PGE1 had no effect on ICa,L, even when either PDE3 or
PDE4 was blocked. However, concomitant inhibition of PDE3 and PDE4 increased ICa,L in
the presence of PGE1, but this effect was not different from the effect of the inhibitors on the
basal ICa,L (Fig. 7D). These results confirm the specific and functional coupling of Glu-R to
PDE4, and show that the cAMP compartment generated by activation of PGE1-R is not in
contact with L-type Ca2+ channels.
(Figure 6 and 7 near here)
Discussion
While a number of recent studies have underscored the role of PDEs in tailoring hormonal
cAMP signals,9-13,21-23 none has addressed directly whether distinct hormonal cAMP signals
are controlled by different PDEs. To gain some insight into this question, we used complementary biochemical and electrophysiological techniques to resolve cAMP at the
cellular, subsarcolemmal and local levels. We show that four canonical GsPCRs expressed in
ARVMs generate different cAMP signals due to a specific contribution of individual PDE
families. For all receptors but PGE1-R, the nature of this functional coupling determines the
regulation of a major downstream cAMP target, the L-type Ca2+ channels.
In agreement with previous studies, we found that PDE3 and PDE4 represent the
major cAMP hydrolytic activities in rodent cardiomyocytes.12,13,24 While there is a general
agreement that β1-AR increases cAMP in adult rat cardiac preparations,3,25-27 the ability of β2
-AR to do so is a matter of debate.3,25,26 Although glucagon increases cAMP in various cardiac
preparations,4 recent studies failed to detect it.27,28. Thus, we checked whether stimulation of
the different receptors led to detectable increases in total cAMP in ARVMs. We found that
stimulation of β1-AR, Glu-R, and to a lesser extent β2-AR, increased total cAMP content, and
that IBMX greatly accentuated these effects. In contrast, PGE1 alone did not increase cAMP,
although a small effect could be observed in the presence of IBMX. This result is at variance
with those from Buxton & Brunton29 in rabbit cardiomyocytes, where ISO and PGE1 similarly
increased cAMP. However, in addition to species differences, the experimental conditions
used here are different, as PGE1 was used at a 10-fold lower concentration (1 vs. 10 µmol/L)
and application lasted 5-fold less (3 vs. 15 min).
We next used CNG channels to monitor cAMP signals triggered by distinct routes
near the plasma membrane.11,13 We found that activation of β1-AR and Glu-R elicit readily
detectable cAMP at the membrane, whereas β2-AR and PGE1-R do not. In heterologous
expression systems and in rat olfactory epithelium, CNG channels associate preferentially
with non-caveolar lipid rafts.30 If this holds true in ARVMs, failure to detect cAMP elicited
by β2-AR and PGE1-R could be due to localization of these receptors in caveolae.31-34
However, this localization does not prevent cAMP diffusion to CNG channels when PDEs are inhibited Indeed, we found that IBMX, at 100 µmol/L concentration, increased the response
of ICNG not only to β1-AR and Glu-R stimulation, but also revealed cAMP generated by β2
-AR and PGE1-R stimulation. Given the Ki of IBMX for the different PDEs,35 100 µmol/L of
this inhibitor should block most of the PDE activity, PDE1, PDE2, PDE3 and PDE4 being inhibited by ≈98%, ≈93%, ≈97% and ≈87%, respectively. To delineate the specific contribution of PDE4, we used Ro 20-1724, which at 10 µmol/L should inhibit ≈71% of its
activity without affecting the other PDEs.35 We found that PDE4 hydrolyzes cAMP produced
by all the receptors tested, thus limiting PKA activation and Ca2+ channel phosphorylation
upon β1-AR, β2-AR and Glu-R activation. These results are consistent with previous studies
showing that PDE4 is critically involved in the regulation of ICa,L and contractility by
catecholamines and glucagon.21,28,36 PDE4 isoforms are likely to play this ubiquitous role due
to their molecular diversity and specific subcellular localization.37-39 However, whether
specific PDE4 isoforms are associated with different receptors remains unclear. Recent data in neonatal cardiomyocytes reported an important role for PDE4B2 in controlling
norepinephrine-induced cAMP increase.12 However, in the same model, other studies suggest
that PDE4D isoforms are preferentially associated with the β2-AR. For instance, PDE4D5 and
to a lesser extent PDE4D3 are recruited to the β2-AR by β-arrestin upon ISO challenge.22,40
Moreover, Xiang et al. (2005)16 showed that PDE4D selectively affects β2-AR vs. β1-AR
Nearly complete (≈95%) and selective PDE3 inhibition should be achieved by 1
µmol/L cilostamide35. Using this inhibitor, we found that PDE3 controls the cAMP signals
generated by β1-AR, β2-AR and PGE1-R, but not Glu-R. However, on ICa,L response, PDE3
controlled the effects of β1-AR and β2-AR, but had no impact on Glu-R or PGE1-R
stimulation. We showed previously that PDE3 controlled cAMP diffusion and consequently
ICa,L activation in cardiac myocytes subjected to β−adrenergic stimulation.13,21,36,41,42 Our
findings contrast with results obtained in neonatal rat ventricular myocytes where PDE3 was
found to have a moderate effect on β1-AR (with norepinephrine) induced cAMP.12 Moreover,
in right ventricular strips from adult rat, PDE3 inhibition had no effect on the contractile
response to the β1-AR agonist dobutamine but potentiated that of glucagon.27 In parallel
cAMP assays, these authors found that PDE3 inhibition had also no effect on the response to dobutamine but revealed a substantial cAMP increase caused by glucagon. While obvious differences in developmental stage, cardiac territory or rat strains might account for some of the discrepancies, one should emphasize that CNG channels provide a readout of subsarcolemmal cAMP, while other methods measure cytosolic or total cAMP. Thus, one
could speculate that upon β1-AR stimulation, PDE3 controls the membrane cAMP pool but
not a cytosolic one, possibly involved in contractility (sarcoplasmic reticulum-associated
PDE3 for instance43). In the case of Glu-R, the opposite would be true: PDE3 would not
control subsarcolemmal cAMP generated by Glu-R, but would prevent the access of the second messenger to other, non sarcolemmal inotropic targets. For such a hypothesis to be valid, one must further speculate either that totally segregated cAMP pathways are triggered by the different receptors (including PKA targets), or that differential regulation of PDE3 is triggered by receptor activation. The latter mechanisms was found in frog heart but not in
rat.44
While inhibition of either PDE3 or PDE4 potentiated the β1-AR response, and
selective inhibition of PDE4 only potentiated the response to Glu, concomitant inhibition of
both low Km cAMP PDEs was necessary to observe a substantial cAMP accumulation upon
β2-AR and PGE1-R stimulations. This indicates that both PDE3 and PDE4 are functionally
associated with β2-AR and PGE1-R, but that one is sufficient to lower cAMP below the
detection threshold of CNG channels. When examining the regulation of ICa,L by β2-AR,
single inhibition of PDE3 or PDE4 potentiated the effect of ISO+CGP. This may reflect the ≈3-10-fold higher sensitivity of PKA vs. CNG channels to cAMP. In contrast, single
inhibition of PDE3 or PDE4 failed to reveal any effect of PGE1 on ICa,L. Although
concomitant inhibition of both PDEs readily stimulate ICa,L in these cells and may mask a
PGE1 effect (Fig. 7D), the data suggest that PGE1-R lie in a distinct compartment from L-type
Ca2+ channels.
Upon stimulation of β1-AR, β2-AR, and Glu-R, inhibition of PDE1-4 by IBMX had a
similar effect on ICNG as did concomitant PDE3 and PDE4 inhibition. This suggests that no
other PDE, besides PDE3 and PDE4, played a significant role under our experimental
conditions. However, a very different situation occurred with PGE1: in this case, inhibition of
PDE1-4 by IBMX had a greater effect than concomitant inhibition of PDE3 and PDE4. Thus,
other PDEs, most likely PDE1 or PDE2, may also regulate PGE1-R-mediated intracellular
cAMP signals.
Although this study was performed on healthy cardiac myocytes, it is tempting to speculate that changes in the organization of the cAMP compartments might take place in pathological conditions, such as heart failure. Cardiac hypertrophy and failure are associated
with a profound remodeling of the cAMP signaling pathway in cardiomyocytes45 As to
decreased while PDE4D was unchanged.46 In the same model, a modest reduction in total PDE activity was found in the left ventricular subendocardium but not in the epicardium,
suggesting regional differences.47 In human, sarcoplasmic reticulum-associated PDE3 activity
was shown to be unchanged in heart failure,43 although total PDE3 activity appears reduced
and PDE3A protein expression down-regulated in end-stage failing human heart.48 In rat, the
expression and activity of PDE3 and PDE4 are strongly augmented in hypertension-induced
hypertrophy.49 Finally, a recent study shows that PDE4D deficiency favours ryanodine
receptors aberrant phosphorylation and promotes heart failure in mice.50 Although more work
is needed to obtain a clear picture of the PDE rearrangement occurring in hypertrophy and heart failure, this study suggests that PDE alteration may affect cAMP compartmentation, leading to untargeted hormonal cAMP signals, aberrant phosphorylation of targets proteins and, in fine, contribute to cardiac dysfunction.
Acknowledgements
We thank Patrick Lechêne (INSERM U769) for skilful technical assistance and Dr. Frank Lezoualc’h for critical reading of the manuscript. This work was supported by an INSERM fellowship (to M.C.), Fondation de France (to G.V.), French Ministry of Education and Research (F.R. and A.A.-G.), Association Française contre les Myopathies (F.R.), National Institutes of Health (RO1 HD 20788) (to M.C. and K.H.) and by European Union Contract n°LSHM-CT-2005-018833/EUGeneHeart (to R.F.).
Conflict of interest statement
None.
Figure legends
Figure 1. Relative PDE activities in ARVMs. PDE2 activity was determined as the fraction of total hydrolytic activity inhibited by 10 µmol/L EHNA; PDE3 as the fraction inhibited by 1 µmol/L cilostamide; and PDE4 activity as the fraction inhibited by 1 µmol/L rolipram. To some instances, rolipram was replaced by RS25344 (1 µmol/L) or piclamilast (1 µmol/L) with identical results. The bars represent the mean±s.e.m of the number of independent experiments indicated.
Figure 2. Effect of various stimuli linked to adenylyl cyclase activation on total cAMP
content in freshly isolated ARVMs. Where necessary, the β1-AR antagonist CGP 20712A
(CGP, 1 µmol/L), the β2-AR antagonist ICI 118551 (ICI, 1 µmol/L) and IBMX (100 µmol/L)
were added 15 min before the 3 min stimulation with ISO (5 µmol/L), glucagon (Glu, 1
µmol/L) or PGE1 (1 µmol/L). The bars show the means±s.e.m. of the number of experiments
indicated near the bars. Statistically significant difference in cAMP content between basal and agonists alones or between IBMX and agonists with IBMX are indicated as ***, p<0.001.
Figure 3. Subsarcolemmal cAMP signals reported by CNG channels upon activation of
distinct GsPCRs in ARVMs. CNG current (ICNG) density from recombinant E583M (A) and
C460W/E583M (B) CNGA2 channels was measured by the whole-cell patch-clamp technique
in rat ventricular myocytes 24 h after isolation. Activation of β1-AR was achieved by
application of isoprenaline (ISO, 5 µmol/L) in the presence of the ICI 118551 (ICI, 1
µmol/L). Activation of β2-AR was done by application of ISO (5 µmol/L) in combination
with CGP 20712A (CGP, 1 µmol/L). Activation of Glu-R (C) and PGE1-R (D) was done by
Figure 4. PDE regulation of cAMP signals from β1-AR and β2-AR. A and C, Time course of
ICNG in ARVMs expressing C460W/E583M CNGA2. The cells were first superfused with
control external Ringer and then challenged with the drugs during the periods indicated by the solid lines. B and D, Summary of the results obtained in a series of experiments as in A and
C, respectively. Specific activation of β1-AR and β2-AR as in Fig. 1. Selective PDE3
inhibition by cilostamide (Cil, 1 µmol/L) or selective PDE4 inhibition by Ro 20-1724 (Ro, 10
µmol/L) strongly potentiated cAMP triggered by β1-AR. Upon activation of β2-AR, robust
cAMP accumulation necessitated concomitant PDE3 and PDE4 blockade. Experiment was ended with application of 100 µmol/L L-85. .Statistically significant differences as in figure 3.
Figure 5. PDE regulation of cAMP signals from Glu-R and PGE1-R. A and C, Time course of
ICNG in ARVMs expressing C460W/E583M CNGA2. B and D, Summary of the results
obtained in a series of experiments as in A and C, respectively. A and B, Cil (1 µmol/L)
exerted a transient potentiation of ICNG previously enhanced by glucagon (Glu, 1 µmol/L),
while Ro (10 µmol/L) exerted a major and sustained cAMP accumulation. C and D, The
cAMP response to PGE1 was revealed at the membrane by simultaneous blockade of PDE3
and PDE4 and further increased by application of IBMX. Statistically significant differences are indicated as *, p<0.05.
Figure 6. Regulation of ICa,L by β-AR and PDEs. A and C, Time course of ICa,L in ARVMs
cultured for 24h. The cells were first superfused with control external Ringer and then challenged with the drugs during the periods indicated by the solid lines. ISO was used at 1 nmol/L in A and B, and at 5 µmol/L in C and D; all other drugs as in previous figures. B and
D, Summary of the results obtained in a series of experiments as in A and C, respectively. Statistically significant differences indicated as in previous figures.
Figure 7. Regulation of ICa,L by Glu−R, PGE1-R and PDEs. A and C, Time course of ICa,L in
ARVMs cultured for 24h. Glucagon (Glu) was used at 1 nmol/L in A and B; all other drugs as in previous figures. B and D, Summary of the results obtained in a series of experiments as in A and C, respectively. Statistically significant differences indicated as in previous figures. NS, non significant.
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CIRCRE SAHA/2006/124800/R1 27
Fi
g
ure
1
Total PDE 2 PDE 3 PD E 4 0 25 50 75 100 125 150PDE i
s
o
for
m
PDE
activ
ity
(p
mole
s/mi
n/ mg
pro
te
in)
(4) (3 ) (3 ) (4)CIRCRE SAHA/2006/124800/R1 28
IB
M
X
Fi
gure 2
ISO
+
IC
I
ISO
+
CGP
Gl
u
P
GE1
IS
O
+
IC
I
IS
O
+
CG
P
Gl
u
P
GE1
0 5 10 20 40 60 80 10 0 12 0Total cA
MP
(p
mo
l/mg)
***
***
***
***
***
***
(9 ) (9 ) (9) (9 ) (9 ) (9 ) (8 ) (8 ) (9 ) (9 )CIRCRE SAHA/2006/124800/R1 29 basal ISO + CGP (9 ) (9 ) ba s a l ISO + CGP (9 ) (9 ) ba s a l PG E1 (1 1) (11 ) E5 8 3 M C460 W /E58 3 M 0 10 20 30 IS O + IC I ba sa l (5 ) (5 ) * I CNG density ( pA /pF ) ** * ** * IS O + IC I basal (13 ) (1 3 )
A
0 10 20 30 I CNG de nsit y (p A/pF) basal Glu (12 ) (12 ) basal Glu (7 ) (7 ) **B
ba s a l PGE 1 (6 ) (6 )Figure 3
CIRCRE SAHA/2006/124800/R1 30 0 10 20 30 40 50 60 70 80 IC I ISO Ci l Ro IB M X L-8 5 - - - - - -+ - - - - -+ + - - - -+ + + - - -+ + - + - -- - - - - + + + - - + -I CNG densit y ( pA /p F) 0 10 20 30 40 50 60 70 I CNG densit y ( pA /p F) CG P ISO Ci l Ro IB M X L-8 5 - - - - - -+ - - - - -+ + - - - -+ + + - - -+ + - + - -+ + + + - -- - - - - + + + - - + -(1 1) (1 1) (1 1) (11 ) (11 ) (10 ) (9 ) (9 ) (9 ) (9 ) (7 ) (6 ) (5 ) (6 ) (5 )
**
***
*
*
***
***
0 1 2 3 4 5 6 0 51 0 1 5 2 0 2 5 ISO + ICI Cil R o IBMX L-8 5 I CNG at -50m V (nA ) Ti m e (m in ) Tim e (m in) 0 1 02 0 3 04 0 5 0 6 0 7 0 8 0 9 0 0 2 4 6 8 10 12 14 I CNG at -50m V (nA ) ISO + CGP L-8 5 IBM X Cil Ro Ci l R o 8A
B
C
D
Fi
gure 4
CIRCRE SAHA/2006/124800/R1 31
Fi
gure 5
0 10 20 30 40 50 60 70 PGE 1 Ci l Ro IB M X L-85 - - - - -+ - - - -+ + + - -- - - - + + - - + -I CNG densit y ( pA /p F) (6 ) (6 ) (6 ) (6 ) (5 ) * * 0 10 20 30 40 50 60 70 80 X 5 - - - - -+ - - - -+ + - - -+ - + - -+ + + - -- - - - + + - - + -** * (7) (7) (7 ) (7 ) (7 ) (6 ) (6 ) *C
D
0 2 4 6 8 10 0 1 02 0 3 04 0 5 0 6 0 T ime ( m in ) PGE 1 Ci l + Ro IB M X L-85 I CNG at -5 0 m V ( nA ) 0 1 2 3 4 5 6 7 8 0 1 02 0 3 04 0 5 0 6 0 7 0 8 0 Tim e (m in) 9 Cil Ro Ci l + Ro IBM X L-85 I CNG at -50 m V (nA ) GluCIRCRE SAHA/2006/124800/R1 32 Time (min) I Ca, L (% ofcont rol ICI ) 10 0 12 0 14 0 16 0 18 0 20 0 (1 1 ) (9 ) (9 )
IC
I
Ci
l
Ro
IS
O
+
-
-+
+
-+
+
+
-+
+
45 Ti me (mi n) 2.5 2.0 1.5 1.0 0.5 0 0 5 10 15 20 25 30 35 40 I Ca,L amplitude (n A) ICI ISO CiI Ro IS O CGP 0 51 0 1 5 20 Ci l Ro 0. 5 1. 0 1.5 2. 0 2.5 3.0 0 I Ca,L amplitude (n A)A
B
C
D
**
CGP
Ci
l
Ro
ISO
+
+
-
-+
+
+
-+
+
+
-+
+
-
+
10 0 15 0 20 0 I Ca, L (% ofcon tro l CG P) (8 ) (9 ) (1 1 ) 12 5 17 5***
**
Figure 6
CIRCRE SAHA/2006/124800/R1 33 0 0 5 10 1 5 20 25 3 0 T im e ( m in ) Glu Ci l Ro Glu Ci l Ro -+ + + + + -80 10 0 12 0 14 0 16 0 (7 ) (6 ) (6 ) *** I Ca,L ( % of control)
B
T im e (m in ) I Ca, L amplit ude (nA ) 2.5 01 0 2 0 3 0 4 0 0.5 1.0 1.5 2.0 PG E1 Cil + Ro Ci l R o 0 PG E 1 Ro Ci l + - -+ - + + + + + -- + + + I Ca, L (% o fcontrol ) NS 90 11 0 13 0 15 0 17 0 19 0 21 0 23 0C
D
(6 ) (6 ) (5 ) (5 ) (4 ) ** *Figure 7
Material and Methods
Isolation, Culture and Infection of ARVM - The investigation conforms with the European Community guiding principles in the care and use of animals (86/609/CEE, CE Off J n°L358, 18 December 1986), the local ethics committee (CREEA Ile-de-France Sud) guidelines and the French decree n°87/748 of October 19, 1987 (J Off République Française, 20 October 1987, pp. 12245-12248). Authorizations to perform animal experiments according to this decree were obtained from the French Ministère de l'Agriculture et de la Forêt (nº7475, May 27, 1997). Male Wistar rats (160-180 g) were subjected to anesthesia by intraperitoneal injection of pentothal (0.1 mg/g) and hearts were excised rapidly. Individual ARVMs were
obtained by retrograde perfusion of the heart as previously described.1 Freshly isolated cells
were suspended in minimal essential medium (MEM: M 4780; Sigma, St. Louis, USA)
containing 1.2 mmol/L Ca2+, 2.5% fetal bovine serum (FBS, Invitrogen, Cergy-Pontoise,
France), 1% penicillin-streptomycin and 2% HEPES (pH 7.6) and plated on laminin coated
culture dishes (10 µg/mL laminin, 2h) at a density of 104 cells per dish. The cells were left to
adhere for 1 hour in a 95% O2, 5% CO2 incubator at 37°C, before the medium was replaced
by 400 µL of FBS-free MEM. For experiments with CNG channels, the E583M or the C460W/E583M CNGA2-encoding adenovirus were added to 200 µL of MEM at a multiplicity of infection (MOI) of 3000 pfu/cell. After 2 hours, the same volume of FBS-free medium without adenovirus was added and the cells were placed overnight in incubator. The medium was changed the next morning for adenovirus- and FBS-free MEM. Patch-clamp experiments were performed the same day.
Electrophysiological Experiments - The whole-cell configuration of the patch-clamp
technique was used to record the L-type calcium current (ICa,L) and the CNG current (ICNG), as
previously described.2 For I measurement, the cells were depolarized every 8 seconds to 0
potassium currents were blocked by replacing all K+ ions with external and internal Cs+. For
ICNG measurement, the cells were maintained at 0 mV holding potential and routinely
hyperpolarized every 8 seconds to –50 mV test potential during 200 ms. The 0 mV holding
potential was chosen because it corresponds to the reversal potential of ICNG under our
experimental conditions. ICNG was recorded in the absence of divalent cations in the
extracellular solution allowing monovalent cations to flow through the channels in an unspecific manner. All the experiments were done at room temperature (21-27°C), and the temperature did not vary by more than 1°C in a given experiment.
Solutions and Drugs for Patch-clamp Recording - Control zero-Ca2+/Mg2+ extracellular Cs+
Ringer solution contained (in mmol/L): NaCl 107.1, CsCl 20, NaHCO3 4, NaH2PO4 0.8,
D-Glucose 5, sodium pyruvate 5, HEPES 10, adjusted to pH 7.4 with NaOH. For ICNG recording,
this solution was supplemented with nifedipine (1 µmole/L) to block nonspecific cation
current through L-type Ca2+ channels. For ICa,L recording, nifedipine was omitted and 1.8 mM
CaCl2 and 1.8 mM MgCl2 were added to the external solution. Control and drug-containing
solutions were applied to the exterior of the cell by placing the cell at the opening of a 250 µm inner diameter capillary tubing. Patch electrodes (0.8-1.2 MΩ) were filled with control
internal solution containing (in mmol/L): CsCl 118, EGTA 5, MgCl2 4, sodium
phosphocreatine 5, Na2ATP 3.1, Na2GTP 0.42, CaCl2 0.062 (pCa 8.5), HEPES 10, adjusted to
pH 7.3 with CsOH. Isoprenaline (ISO), prostaglandin E1 (PGE1), glucagon, ICI 118551 (ICI),
CGP 20712A (CGP), rolipram, erythro-9-(2-hydroxy-nonyl)adenine (EHNA) and 3-isobutyl-1-methyl-xanthine (IBMX) were purchased from Sigma. L-858051 (L-85, a hydrosoluble analogue of forskolin) and cilostamide were purchased from France Biochem (Meudon, France). Ro 20-1724 (Ro) was kindly provided by Hoffman-La-Roche (Basel,
Alto, CA, USA).
PDE Assay - Freshly isolated ARVMs were seeded on 35 mm Petri dishes at a density of 105
cells/dish in FBS-free medium. After 1h, cells were washed with PBS and homogenized in ice-cold buffer containing (in mmol/L) NaCl 150, sodium phosphate buffer (pH 7.2) 10, EDTA 2, β-mercaptoethanol 5, sodium pyrophosphate 30, sodium fluoride 50, benzamidine 3,
AEBSF 2, NP40 0.1%, leupeptin 5 µg/ml, pepstatin 20 µg/ml, microcystin 1 µM. PDE activity was measured according to a modification of the two-step assay procedure method
described by Thompson & Appleman3 in a total volume of 200 µL including (in mmol/L)
Tris-HCl 40, pH 8.0, MgCl2 10, β-mercaptoethanol 1.25 supplemented with 1 µM cAMP and
105 cpm [3H]-cAMP, as detailed previously.4 PDE family-specific activities were determined
as the difference between PDE activity in the absence of inhibitor and the residual hydrolytic activity observed in the presence of the selective inhibitor. Protein concentration was determined by bicinchonic acid assay after addition of deoxycholate and precipitation by trichloroacetic acid (0.1%).
cAMP Level Determination - Freshly isolated ARVMs suspended in FBS-free MEM were
plated in laminin-coated 12-well plates at a density of 5x104 cells/well. After 1h, the medium
was replaced with control zero Ca2+/Mg2+ extracellular Cs+ Ringer solution in the presence or
absence of β-adrenergic antagonists ICI (1µmol/L) or CGP (1 µmol/L), or IBMX (100
µmol/L). After 15 min., the same Ringer solution with or without receptor agonists (ISO, 1
µmol/L; glucagon, 1 µmol/L; or PGE1, 1 µmol/L) was applied for 3 min at room temperature.
The stimulation was stopped by ice-cold trichloroacetic acid (0.1%) in 95% EtOH. After 30 min incubation on ice, samples were scraped and centrifuged for 30 min at 3,000 rpm at 4°C. The pellet was dissolved in 5% SDS in 0.1 N NaOH for protein determination by bicinchoninic acid assay. EtOH in the supernatant was evaporated and the material was
samples and appropriate dilution.5,6
Results
For each receptor, identical experiments were performed in myocytes expressing the low affinity cAMP sensor E583M CNGA2, and gave essentially similar results. As shown in
supplementary Fig IA and IB, individual inhibition of PDE3 and PDE4 potentiated the β1-AR
response, and these effects were not different from the effect of IBMX. In contrast, simultaneous inhibition of PDE3 and PDE4 was necessary to unmask a substantial and
sustained sarcolemmal cAMP accumulation in response to β2-AR stimulation (supplementary
Fig IC and ID), showing that either PDE3 or PDE4 activity is enough to dampen β2-AR
cAMP at the membrane. As noted above, ICNG was not augmented by cell stimulation with
glucagon when using the low affinity CNG channel (Fig. IIA and IIB). However, similarly to what observed with the C460W/E583M mutant, selective blockade of PDE3 failed to increase
ICNG while PDE4 inhibition induced a major rise in ICNG. Supplementary Fig. IIC and IID
present the results obtained when PGE1 was used to trigger cAMP in cardiac myocytes. While
the hormone alone failed to increase ICNG, the additional and concomitant inhibition of PDE3
and PDE4 provoked a slow accumulation of cAMP at the membrane. This accumulation was enhanced when all PDEs were inhibited with IBMX (supplementary Fig. IIC and IID).
Legends to Supplementary Figures
Supplementary Figure I. PDE regulation of cAMP signals from β1-AR and β2-AR. A and C,
a series of experiments as in A and C, respectively. Specific activation of β1-AR and β2-AR as
in Fig. 1. Cilostamide (Cil, 1 µmol/L) was used for specific inhibition of PDE3 and Ro 20-1724 (Ro, 10 µmol/L) for specific inhibition of PDE4. IBMX (100 µmol/L) was used for non specific inhibition of PDEs. At the end of the experiment, the cell was challenged with a saturating concentration of the forskolin analog L-858051 (L-85, 100 µmol/L) as an internal control for CNG channels expression. The bars show the means±s.e.m. of the number of cells indicated. Statistically significant differences are indicated as **, p<0.01 and ***, p<0.001.
Supplementary Figure II. PDE regulation of cAMP signals from Glu-R and PGE1-R. A and
C, Adult rat ventricular myocytes infected with E583M Ad-CNG for 24h were superfused for a few minutes with a control Ringer solution and then challenged with a drug during the periods indicated with the solid lines. B and D, Summary of the results obtained in a series of experiments as in A and C, respectively. Glu-R activation was achieved by application of glucagon (Glu, 1 µmol/L). Pharmacological inhibition of PDEs as in Fig.2. Experiment was terminated with application of 100 µmol/L L-85. The bars show the means±s.e.m. of the number of cells indicated. Statistically significant differences are indicated as *, p<0.05; **, p<0.01 and ***, p<0.001. NS, non significant.
Supplementary references
1-Verde I, Vandecasteele G, Lezoualc'h F, Fischmeister R. Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca2+ current in rat ventricular myocytes. Br J Pharmacol. 1999;127:65-74.
cAMP phosphodiesterase on subsarcolemmal cAMP signals in intact cardiac myocytes: an in vivo study using adenovirus-mediated expression of CNG channels. J Biol Chem. 2004;279:52095-105.
3-Thompson WJ, Appleman MM. Multiple cyclic nucleotide phosphodiesterase activities from rat brain. Biochemistry. 1971;10:311-6.
4-Oki N, Takahashi SI, Hidaka H, Conti M. Short term feedback regulation of cAMP in FRTL-5 thyroid cells. Role of PDE4D3 phosphodiesterase activation. J Biol Chem. 2000;275:10831-7.
5-Harper JF, Brooker G. Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2'0 acetylation by acetic anhydride in aqueous solution. J Cyclic Nucleotide Res. 1975;1:207-18.
6-Steiner AL, Pagliara AS, Chase LR, Kipnis DM. Radioimmunoassay for cyclic nucleotides. II. Adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate in mammalian tissues and body fluids. J Biol Chem. 1972;247:1114-20.
A
B
70 pF) 60 50 40 30 density (pA/ 20 10CNG I 0ICI ISO Cil Ro IBMX L-85
- - - - - -+ - - - - -+ + - - - -+ + + - - -+ + - + - -- - - - - + + + - - + -(5) (5) (5) (5) (5) (5) (5)
**
**
**
CGP ISO Cil Ro IBMX L-85
- - - - - -+ - - - - -+ + - - - -+ + + - - -+ + - + - -+ + + + -
-***
***
I CNG density (pA/ pF) (9) (9) (9) (6) (6) (7) ISO + CGP Cil + Ro IBMX L-85 12 10 8 6 4 2 0 0 1 0 2 0 3 04 05 Time (min) I CNG at -50 mV (nA)D
C
0 10 20 30 40 50 60 9 8 7 6 5 4 3 2 1 0 01 0 2 0 3 0 4 0 50 60 ISO + ICI Cil Ro IBMX CNG Time (min) L-85Suppl
Ro Cil Glu Time (min) 1 0 2 03 0 4 05 0 6 0 7 0 8 0 9 0 Cil + Ro IBMX L-85
